Keiko Hosohata

Purification and Identification of a Novel Complex Which Is Involved in Androgen Receptor-Dependent Transcription

ABSTRACT The androgen receptor (AR) binds to and activates transcription of target genes in response to androgens. In an attempt to isolate cofactors capable of influencing AR transcriptional activity, we used an immunoprecipitation method and identified a 44-kDa protein, designated p44, as a new AR-interacting protein. p44 interacts with AR in the nucleus and with an androgen-regulated homeobox protein (NKX3.1) in the cytoplasm of LNCaP cells. Transient-transfection assays revealed that p44 enhances AR-, glucocorticoid receptor-, and progesterone receptor-dependent transcription but not estrogen receptor- or thyroid hormone receptor-dependent transcription. p44 was recruited onto the promoter of the prostate-specific antigen gene in the presence of the androgen in LNCaP cells. p44 exists as a multiprotein complex in the nuclei of HeLa cells. This complex, but not p44 alone, enhances AR-driven transcription in vitro in a cell-free transcriptional system and contains the protein arginine methyltransferase 5, which acts synergistically with p44 to enhance AR-driven gene expression in a methyltransferase-independent manner. Our data suggest a novel mechanism by which the protein arginine methyltransferase is involved in the control of AR-driven transcription. p44 expression is dramatically enhanced in prostate cancer tissue compared with adjacent benign prostate tissue.

Endomorphin-1 and endomorphin-2 are partial agonists at the human μ-opioid receptor

Recently two tetrapeptide ligands that bind preferentially to the mu-opioid receptor were identified and named endomorphin-1 and endomorphin-2. We examined the ability of these peptides to stimulate G protein activation in human mu-opioid receptor transfected B82 fibroblasts as measured by [35S]GTPgammaS binding to cell membranes. Both endomorphin-1 and -2 act as partial agonists in this assay system compared with the mu-selective agonist [D-Ala2,N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). In addition, endomorphins demonstrate efficacy similar to morphine. These findings demonstrate that endomorphin peptides have similar activity at the mu-opioid receptor as morphine and suggest that these peptides have the potential to modulate neuronal activity in vivo.

AM630 IS A COMPETITIVE CANNABINOID RECEPTOR ANTAGONIST IN THE GUINEA PIG BRAIN

AM630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [35S]GTPgammaS binding suggesting that difference in AM630 activity in different tissues is not due to species variation.

Relative efficacies of δ-opioid receptor agonists at the cloned human δ-opioid receptor

The present study was conducted to determine the relative efficacies of the selective δ-opioid receptor agonists SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[d-Pen2,4′-ClPhe4,d-Pen5]enkephalin) and (−)-TAN67 ((−)-2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPγS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human δ-opioid receptor. Efficacy was determined according to the formula: efficacy=(Emax-A/Emax)(A′/A+1)×0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6–7 times greater than that of (−)-TAN67.

The role of the G protein γ2 subunit in opioid antinociception in mice

Abstract We examined the role of the γ2 subunit of G proteins (Gγ2) in the antinociception produced by c[D-Pen2,D-Pen5]enkephalin (DPDPE) in mice. DPDPE produced 84.0±9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Gγ2 subunit, DPDPE-mediated antinociception decreased to 24.4±7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1±10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4±23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P

(2S,3R)TMT-l-Tic-OH is a potent inverse agonist at the human δ-opioid receptor

Abstract We examined the pharmacologic effect of β-methyl-2′,6′-dimethyltyrosine- l -tetrahydroisoquinoline-3-carboxylic acid ((2S,3R)TMT- l -Tic-OH) on G protein activation in membranes prepared from Chinese Hamster Ovary cells transfected with cDNA of the human δ-opioid receptor. (2S,3R)TMT- l -Tic-OH inhibited G protein activation to 58% of basal with an EC50 of 0.72 nM as determined by [ 35 S ]GTPγS binding. These findings suggest that (2S,3R)TMT- l -Tic-OH is a highly potent inverse agonist at the human δ-opioid receptor.

The efficacy of δ-opioid receptor-selective drugs

Delta-opioid receptor-selective drugs may provide an alternative to mu-opioid-selective drugs currently used for the relief of pain. To develop improved delta-opioid receptor-selective drugs, better measures of drug activity are necessary. In this review we suggest that efficacy calculations provide a superior measure of drug activity as compared to dissociation constants and drug potencies in functional assays. Efficacy, as discussed in this review, is defined as a quantitative measurement of the ability of a drug to stimulate second messenger systems or measurable functional responses in cells or tissues under standard conditions. Efficacy values will allow medicinal chemists to understand the contributions of both the coupling efficiency and dissociation constant to drug potencies in the development of new delta-opioid receptor-selective drugs.

Biological properties of Phe°-opioid peptide analogues

Biological properties of new analogues, which represent Phe(o)-propeptides of a variety of opioid peptides, are described. All Phe(o)-opioid analogues expressed both receptor binding affinities and in vitro biological activities at least at the level of the primary opioid peptides. Surprisingly, some of the propeptides expressed slightly higher activity than the primary opioid peptides. Nevertheless, no significant shift in receptor selectivity was observed, which indicate that these Phe(o)-analogues undoubtedly are propeptides. The possible role of membrane proteolytic enzymes associated with opioid receptors in transformation of propeptides is discussed.

Design and synthesis of nanomolar δ-opioid selective nonpeptide mimetic agonists based on peptide leads

The synthesis of designed non-peptide mimetics began with commercially available 3hydroxylbenzaldehyde 1 as shown in Scheme 1. The free hydroxyl group was protected as a methoxyethoxymethyl ether 2 which was reacted with various Grignard reagents or reduced by sodium boron hydride to form the secondary and primary alcohols 3, respectively. The intermediate 3 was converted into chloride 4 in the presence of triphenylphosphine and tetrachloromethane. The chloride 4 was then reacted with 1benzylpiperazine to form intermediate 5 whose hydroxyl protection were removed with 2N hydrochloric acid to provide the desired products 6 as hydrochloride acid salts. The binding assay results of the synthesized non-peptide mimetics against radio labeled highly selective opioid ligands are listed in the Table 1. As shown, the hydrophobicity of the R group has a dramatic effect on the binding and selectivity of the non-peptide ligand 6 to the receptor. Analogue 6e (SL 3111) with a very hydrophobic para-t-butylphenyl substituent showed the highest binding affinity nM) with over 2000 fold greater selectivity for the receptor than for the receptor. This exceeds the selectivity of the peptide lead for the opioid receptor. Although a further in vitro bioassay on 6e (SL 3111) indicates that its in vitro bioactivity with an nM at MVD and its selectivity are lower than expected. This may be due to the racemic materials examined. Alternatively, the ligand does not have all the proper pharmacophores to interact with the receptor. Nonetheless, SL-3111 is a novel selective non-peptide lead that indeed mimics the peptide ligand. Further modification on this non-peptide lead is currently underway in our laboratory.

Design of a δ opioid peptide mimetic based on a topographically constrained cyclic enkephalin

A major goal in modern drug research is the ability to design de novo a non-peptide mimetic of the pharmacophore of a conformationally constrained receptor selective peptide hormone or neurotransmitter agonist for a 7-transmembrane G-linked receptor with high potency, selectivity and agonist efficacy. Thus far success has been very limited, since there is still much to learn about the structure, conformation, topography, and dynamics related to agonist activity at receptors [1]. For example, it is not clear that any of the many putative non-peptide structure mimetics or scaffolds [e.g. 2, 3] for peptide secondary structure (β-sheets, β-turns, etc.) actually can mimic the specific stereoelectronic, topographic and dynamic (e.g. α-helix-coil-β-sheet interconversion) properties of a peptide that may be critical in their agonist biological activities. On the other hand, a great deal of success has been realized in designing peptidomimetics that, although greatly modified structurally and topographically from the natural peptide agonist ligand, nonetheless retain many of the stereoelectronic and functional groups of peptides [e.g. 4, 5], as well as their agonist activities. Indeed, it is not clear what kinds of non-peptide scaffolds can be used to design ligands that can mimic the secondary structure, topography, and stereoelectronic properties of a peptide necessary for agonist activity. To help evaluate which conformationally flexible scaffolds may be used to mimic peptide agonist pharmacophores, we have used a highly potent, receptor selective, conformationally and topographically constrained peptidomimetic, computational chemistry and molecular modeling to design a non-peptide ligand that could mimic the major proposed pharmacophore elements. Based on these studies we have designed and synthesized a non-peptide mimetic of a δ receptor selective opioid ligand with high potency and selectivity similar to the peptide ligand on which it was based, but with low efficacy.